The cellular cytoskeleton functions to maintain the spatial shape and volume of mammalian cells; it is crucial for functions such as locomotion (migration), endocytosis, phagocytosis, and mechanoprotection. Actin is one of the key proteins of the cytoskeleton, and the physiologic assembly and disassembly of actin filaments in response to regulatory signals maintains homeostasis of cellular functions and responds to external stresses to protect the cell. Actin filaments are often found in bundles called stress fibers that terminate in focal complexes at the plasma membrane, where they communicate with the extracellular matrix through focal adhesions and associated membrane proteins called integrins. Cells move by remodeling actin filaments at their leading edge. Inflammatory cells like neutrophils and macrophages require actin filament assembly to migrate to sites of infection or injury and to engulf microorganisms or other foreign objects. Cells like fibroblasts that maintain the integrity of connective tissue extracellular matrix (ECM; collagen, for example) require actin filament assembly to migrate to heal wounds and to phagocytose collagen to maintain homeostasis of the ECM. Epithelial cells require actin filament assembly to migrate over wounds and to maintain cell-cell junctions in a protective barrier that protects the underlying connective tissue from microbes and their products. So, the cytoskeleton is a central part of the cellular machinery in virtually all cells and all tissues of the body.
Phalloidin, jasplakinolide and amphidinolide H are three actin-stabilizing agents that bind to actin filaments and aggregate them in vitro (Holzinger and Meindl, 1997; Lee et al., 1998; Saito et al., 2004). Besides their application in investigating actin dynamics and actin dependant events in mammalian cells, phalloidin has become a very valuable reagent in cytoskeletal imaging techniques when conjugated to fluorescent dyes. Similar to phalloidin, Jasplakinolide induces actin polymerization and stabilizes pre-existing actin filaments in vitro (Bubb et al., 1994, Holzinger, 1997; Mathur et al., 1999; Rosado and Sage, 2000, Bubb et al., 2000). Jasplakinolide differs from phalloidin, in that it is cell permeant. It gets into the cell and can therefore be more easily used for in vitro experiments using cells in culture. Jasplakinolide is a naturally occurring cell permeable cyclic peptide, produced by a sponge, Jaspis johnstoni. It is known to inhibit the growth of prostate carcinoma cell lines in vitro (Senderowicz et al., 1995), and to prevent the self-renewal of acute myeloid leukemia cells (Fabian et al., 1995). In each of these reports, the effects of Jasplakinolide were attributed to its ability to bind and stabilize actin filaments.
There are a number of laboratory reagents, like Cytochalasin, that are highly useful in research because they are cytoskeletal inhibitors, but there are fewer laboratory reagents with cytoskeletal stabilizing effects. The above-noted actin-stabilizing reagents come from living organisms: mushrooms, sponges, and algae. Obtaining such agents from biological sources involves labour-intensive procedures. Thus, the development of new synthetically accessible laboratory reagents with cytoskeletal stabilizing effects is highly desirable.
Over the past 15 years, there has been a great expansion of knowledge about how bacteria exploit or perturb the host cell cytoskeleton. Numerous bacterial proteins that act on cytoskeleton-regulating signal transduction pathways have been identified and characterized (Finlay and Cossart, 1997; Cossart, 1997; Cossart and Lecuit, 1998; Aktories and Barbieri, 2005; Patel and Galan, 2005; Rottner et al., 2005; Caron et al., 2006). Notably, bacterial agonists of cytoskeletal remodeling are now being considered for their potential applications in modulating membrane receptor activity and innate immunity, concentrating mostly on bacteria that are exogenous pathogens (Finlay and Hancock, 2004; Hayward et al., 2006). The same strategies applied to indigenous bacteria, which have evolved in concert with the host, would theoretically expand the probability of identifying potential target molecules.
Cellular turnover among gingival epithelial cells and ECM turnover in the periodontal connective tissues are among the most rapid in the body due to these tissues being in constant proximity to a large mass of bacteria that colonize on the teeth as a subgingival biofilm. Treponema denticola is one of the most prominent bacteria that colonize at the interface of the bacterial biofilm and the gingival tissues, and it is a key organism associated with progressive chronic periodontitis (Ellen and Galimanas, 2005).
The major surface antigen of T. denticola is known as the major outer sheath protein (Msp). This protein was first characterized in McBride's laboratory at the University of British Columbia (Haapasalo et al, 1992). The full gene was cloned, sequenced, and investigated in detail, as reported in subsequent publications (Fenno et al., 1996, 1997). For over a decade, Applicants have investigated the effects of T. denticola and more recently Msp on oral host cells, mostly gingival fibroblasts and epithelial cells, and recently neutrophils. Applicants' laboratory is the only laboratory that has reported on the cytoskeletal effects of Msp and of this bacterium.
Current literature (Amin et al., 2004; Batista da Silva et al, 2004, Wang et al., 2001) suggests that the major outer sheath protein (Msp) of the indigenous oral spirochete Treponema denticola induces significant cytoskeletal reorganization through disassembly of actin filaments and stress fibers ventrally near the middle of the cell, accompanied by the assembly of a mesh of subcortical actin filaments that disrupt some cellular functions. A few recent papers that are highly novel show that cell exposure to Msp uncouples store operated calcium channels (SOCs) and prevents the normal affinity modulation (cellular adhesion, spreading, and migration) when fibroblasts contact extracellular collagen (see FIG. 1). Both effects appear to involve Msp-induced reorganization of the actin cytoskeleton, including assembly of a subcortical mesh of actin filaments. These novel discoveries are published in Wang et al. 2001, Paes da Silva et al. 2004, and Amin et al. 2004 and references therein. Msp also inhibits chemotaxis in neutrophils and suppresses their calcium and actin responses to chemoattractants (Puthengady Thomas et al., 2006). Since these publications, Applicants have been trying to seek ways to determine the active peptide domains of Msp that determine its cytoskeletal effects.
During the course of these studies, Applicants have surprisingly and unexpectedly produced a novel peptide conjugate with cytoskeletal stabilizing effects.